CN103525773B

CN103525773B - A kind of method and application thereof improving PRRS virus target cell infection titre

Info

Publication number: CN103525773B
Application number: CN201310491696.8A
Authority: CN
Inventors: 黄海军
Original assignee: WUHAN INST OF VETERINARY SCIENCE
Current assignee: WUHAN INST OF VETERINARY SCIENCE
Priority date: 2013-10-18
Filing date: 2013-10-18
Publication date: 2015-11-18
Anticipated expiration: 2033-10-18
Also published as: CN103525773A

Abstract

The invention discloses a kind of method and the application thereof that improve PRRS virus target cell infection titre, belong to animal epidemic prevention and control field.By building the single or multiple Marc145 cells or MA-104 cell that can express in PRRS virus acceptor Suleparoid, sialoadhesin, vimentin, CD163, CD151 and non-muscle myoglobulin heavy chain II A acceptor, recycle this cell Pigs Inoculated reproductive and respiratory syndrome virus, collect virus liquid, PRRS virus infection titer is largely increased; The virus liquid collected can be further used for blue-ear disease vaccine and produce.The present invention utilizes and can express PRRS virus recipient cell virus inoculation, virus can be gathered in the crops continuously and results virus titer raising 2 ~ 5 times, and the effect of prepared vaccine does not reduce, on the basis not increasing extra production cost, unit time production capacity is made to improve 2 ~ 5 times.

Description

A kind of method and application thereof improving PRRS virus target cell infection titre

Technical field

The present invention relates to animal epidemic prevention and control field, particularly relate to and a kind ofly improve the method for PRRS virus target cell infection titre and the application in production of vaccine thereof.

Background technology

Pig blue-ear disease, also porcine reproductive and respiratory syndrome (Porcinereproductiveandrespiratorysyndrome is, PRRS), by porcine reproductive and respiratory syndrome virus (Porcinereproductiveandrespiratorysyndromevirus, PRRSV) viral infectious being principal character with Sow abortion and piglet dyspnoea caused, and cause serious immunosuppression.This virus is from 1987 since the U.S. occurs, wide-scale distribution in global swinery, causes huge financial loss to world's pig industry, one of main epidemic disease becoming global scale pig farm, is also a great problem on global swine disease controls.

Virus receptor is the important factor determining virus host range and tissue tropism.One of the focus and difficult point of current virological investigation about the research of relation between virus receptor specificity and viral origin host type.The prerequisite that PRRS virus infects target cell realizes the absorption with host cell, and the acceptor of host cell surface has been that this adsorption process is requisite.Research shows, the receptors bind on PRRS virus and cell realizes the prerequisite that it infects porcine alveolar macrophage.The cell receptor of the PRRS virus determined at present has six kinds, Suleparoid (HeparinSulphate respectively, HS), sialoadhesin (Sialoadhesin, Sn), vimentin (Vimentin), CD163(ClusterofDifferentiation163) molecule, CD151(ClusterofDifferentiation151) molecule and NMMHCIIA(non-muscle myoglobulin heavy chain II A) molecule.

Along with the development of proteomic techniques, proteomic techniques is more and more important by playing a part in new antiviral drug exploitation.Studied by virus infection Russula subnigricans Hongo, virus function can be screened in the target proteins of cell.Find that the action target of virus resists the exploitation of viral newtype drug significant, can design the antiviral molecule medicines such as polypeptide class with a definite target in view according to the structure of target, blocking virus is to the effect etc. of target.Can the gene of Code targets albumen be transferred in corresponding cellular genome by the means that genetically engineered is modified, strengthen intensity and the time length of cell infection virus, produce the vaccine of more high-titer, the prevention and therapy for virus disease brings unprecedented development space.

Summary of the invention

Primary and foremost purpose of the present invention is that the shortcoming overcoming prior art is with not enough, provides a kind of method improving PRRS virus target cell infection titre.

Another object of the present invention is to provide the application of aforesaid method in pig blue-ear disease vaccine is produced.

Object of the present invention is achieved through the following technical solutions:

Improve a method for PRRS virus target cell infection titre, comprise the steps: to cultivate with cell growth medium the cell can expressing PRRS virus acceptor; When cell fraction of coverage reaches more than 80%, remove cell growth medium, Pigs Inoculated reproductive and respiratory syndrome virus, add cell maintenance medium and continue to cultivate; When the cytopathy that virus causes reaches more than 80%, gather in the crops virus liquid, fill into subsequently not containing the cell maintenance medium of virus, after 12h, receive poison again, so repeatedly receive poison 3 ~ 5 times.

The cell of described expressed PRRS virus acceptor preferably can express Suleparoid (HeparinSulphate, HS), sialoadhesin (Sialoadhesin, Sn), vimentin (Vimentin), CD163(ClusterofDifferentiation163), CD151(ClusterofDifferentiation151) and NMMHCIIA(non-muscle myoglobulin heavy chain II A) etc. single or multiple Marc145 cell in acceptor or MA-104 cell.

Preferred, the cell of described expressed PRRS virus acceptor is the Marc145 cell expressing CD151 molecule, CD163 molecule or co expression CD151 and CD163 molecule.

The cell of described expression PRRS virus acceptor prepares preferably by the method comprising following steps: be connected on carrier for expression of eukaryon by the gene fragment of the described acceptor of coding, be transfected in Marc145 cell or MA-104 cell again, obtain the cell of expressing PRRS virus acceptor by G418 and the screening of single cell clone culture method; Described carrier for expression of eukaryon is preferably pIRES2-EGFP.

Described cell growth medium is preferably and contains the DMEM that volumetric concentration is 8% calf serum; Described cell maintenance medium is preferably and contains the DMEM that volumetric concentration is 2% calf serum.

The dosage of described PRRS virus inoculation is preferably 0.0005 ~ 0.002MOI, preferred, and the dosage of PRRS virus inoculation is 0.001MOI.

The application of method in pig blue-ear disease vaccine is produced of above-mentioned raising PRRS virus target cell infection titre.

After a kind of method of producing pig blue-ear disease vaccine comprises the steps: to collect virus liquid according to the method described above, virus liquid is mixed with lyophilized vaccine, makes freeze-dried live vaccine; Or by the ultra-filtration membrane bag concentrated more than 10 times of virus liquid with 100KD molecular weight cut-off, mixed with concentrated virus liquid by inactivator after carrying out deactivation, then obtain purifying antigen with gel chromatography column column chromatography, by antigen and the mixing of oily adjuvant, emulsification is mixed with inactivated vaccine.

Described lyophilized vaccine is preferably the sucrose skimming milk that mass concentration is 5%; Described fire-fighting medium is preferably the formaldehyde solution that mass concentration is 37%; Described oily adjuvant is preferably prepared with 94% white oil, 6%Span-80 and 2% aluminum stearate.

Described virus liquid and the volume ratio of lyophilized vaccine are preferably 1:0.5 ~ 2, are more preferably 1:1; Described inactivator is preferably 1:1500 ~ 2500 with the volume ratio of concentrated virus liquid, is more preferably 1:2000; The volume ratio of described antigen and oily adjuvant is preferably 1:0.5 ~ 2, is more preferably 1:1.

The present invention has the following advantages and effect relative to prior art tool:

The present invention adopts gene clone method from porcine alveolar macrophage clone pig reproductive and respiratory syndrome virus receptor protein gene encode fragment, be inserted into carrier for expression of eukaryon, PRRSV sensitive cells is stably transfected into by liposome-mediated mode, and adopt limiting dilution method, progressively expand numerous from single positive cell clone, set up the clone (strain) of single PRRSV acceptor or multiple PRRSV acceptor genetic modification, after utilizing this cell Pigs Inoculated reproductive and respiratory syndrome virus, significantly can improve virus infection titer, repeatedly can receive poison, and finally guarantee the stability of produced PRRSV vaccine potency.Such as, in embodiment cited by the present invention, utilizing PRRS virus JXA1-R(JXA1) strain infects stable transfection CD151 molecule, CD163 molecule and co expression CD151 and CD163 molecule, can gather in the crops virus continuously and results virus titer raising 2 ~ 5 times, and the effect of prepared vaccine does not reduce.Total appraisal, adopts technology of the present invention, can, on the basis not increasing extra production cost, make unit time production capacity improve 2 ~ 5 times.

Accompanying drawing explanation

Fig. 1 is goal gene CD151 and the CD163 fragment electrophoretic result figure of pcr amplification; Wherein, A:CD151; B:CD163.

Fig. 2 is the electrophoresis result figure that recombinant plasmid pIRES2-EGFP-CD151 and pIRES2-EGFP-CD163 uses SalI and KpnI double digestion respectively; Wherein, A:pIRES2-EGFP-CD151; B:pIRES2-EGFP-CD163.

Fig. 3 is the result figure of Marc145 cell under fluorescence inverted microscope of stable transfection PRRSV receptor protein gene CD151, CD163 and CD151/CD163 cotransfection; Wherein, the Marc145 cell of A: transfection CD151; The Marc145 cell of B: transfection CD163; The Marc145 cell of C:CD151/CD163 cotransfection; In each figure of A, B, C, left figure is bright field, and right figure is fluorescence microscope result.

The expression result figure that Fig. 4 is PRRSV acceptor in the Marc145 cell of untransfected Marc145 cell and transfection; Wherein, A1 and A2 be respectively untransfected with the Marc145 cell of transfection CD151; B1 and B2 be respectively untransfected with the Marc145 cell of transfection CD163; C1 and C2 be respectively untransfected with the Marc145 cell of CD151/CD163 cotransfection; The blue indicator cells core of left figure in each figure, the red indicating target receptor protein of right figure.

Embodiment

Below in conjunction with embodiment and accompanying drawing, further detailed description is done to the present invention, but embodiments of the present invention are not limited thereto.If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art.

The structure of embodiment 1 PRRS virus receptor protein encoding gene carrier for expression of eukaryon and plasmid extraction

(1) goal gene coding region (CDS) design of primers

According to American National Biotechnology Information center (NCBI, NationalCenterforBiotechnologyInformational; Website http://www.ncbi.nlm.nih.gov/) GenBank in the pig CD151(NM_001243865.1 that reported) and CD163(NM_213976.1) gene order, with this section of sequence for template, primerpremier6 and Oligo6 software is utilized to carry out design of primers.Designed primer sequence is in table 1, and primer is synthesized by Shanghai bio-engineering corporation.

The primer sequence of table 1 pig CD151 and the amplification of CD163 gene cDNA

(+) and (-) represents the upstream and downstream primer of amplified fragments respectively; Italic is protectiveness base; Runic is restriction enzyme site.

(2) pcr amplification of goal gene

The cDNA obtained with porcine alveolar macrophage (PAM cell) the total serum IgE reverse transcription of extracting, for template, carries out pcr amplification with the primer in table 1.Adopt KODDNA polysaccharase, PCR reaction system is 50 μ L systems, comprising ddH ₂o10 μ L, dNTPs8 μ L(concentration is 10mM), 2 × Buffer25 μ L, cDNA template 4 μ L, upstream and downstream primer adds 1 μ L respectively, KODDNA polysaccharase 1 μ L.PCR response procedures: 94 DEG C of denaturation 2min; 98 DEG C of sex change 10s, annealing 30s, 68 DEG C extend 1kb/30s, 35 circulations.

(3) detection of goal gene fragment

After PCR reaction terminates, get 10 μ LPCR amplified productions, add 0.5 μ L sample-loading buffer (10 × LoadingBuffer), after mixing, careful point sample is in the sepharose glue hole of 0.7%, with time point 5 μ LDL1kMarker as reference, under 150V voltage conditions, electrophoresis 40min.After electrophoresis terminates, dye 15min in the damping fluid being added with EB, and taking-up clean water washes away the EB on surface, then utilizes G-BOX gel imaging system to observe electrophoresis result, and as shown in Figure 1, CD151 with CD163 fragment conforms to expection size result.

(4) recovery of object fragment, enzyme are cut and purifying

DNA fragmentation is separated through agarose gel electrophoresis, after EB dyeing, under long wavelength ultraviolet lamp, (time is short as far as possible) guarantees that object fragment and other DNA divide out, accurately cut the sepharose containing object fragment, put into the Eppendorf(EP of an aseptic 1.5mL) pipe, utilize DNA glue to reclaim test kit (Axygen, USA) and reclaim target DNA.Step: add 400 μ L sol solutionses, in 60 DEG C of water-bath 5min, flicks EP pipe therebetween, gel is dissolved completely.The sol solution dissolved is transferred to the recovery post that cover has 2mL collection tube, room temperature leaves standstill 2min, 8000r/min, centrifugal 1min, outwell the waste liquid in collection tube, the washings adding 500 μ L washes twice, the centrifugal 1min of last 12000r/min, going to reclaiming post in a clean 1.5mLEP pipe, adding 10 μ LddH ₂o wash-out, places the centrifugal 1min of 5min, 12000r/min in room temperature, is the DNA fragmentation of recovery, for next step endonuclease reaction in collection tube.

Utilize SalI and KpnI restriction endonuclease to reclaiming the CD151(762bp that obtains) and CD163(3333bp) DNA fragmentation carry out double digestion, 20 μ L systems: 1.5 × T3 μ L, BSA2 μ L, DNA14 μ L, SalI0.5 μ L, KpnI0.5 μ L.

After enzyme cuts 5h in 37 DEG C of thermostat containers, carry out the connection that product that purifying (step with DNA glue reclaim) final purification obtains can be used for next step being reclaimed test kit by DNA.

(5) carrier enzyme is cut, reclaims and is connected

20 μ L enzymes cut system: ddH ₂o10 μ L, 1.5 × T3 μ L, BSA2 μ L, pIRES2-EGFP carrier 4 μ L(2 μ g), SalI0.5 μ L, KpnI0.5 μ L.

After enzyme is cut, whole sample is added to electrophoresis in the sepharose loading wells of 0.7%.Then utilize glue to reclaim test kit to reclaim (method is the same) object fragment.Finally the linear carrier obtained is connected respectively with goal gene DNA, 20 μ L linked system: ddH ₂o9 μ L, buffer2 μ L, DNA6 μ L(0.3pmol), carrier 2 μ L(0.03pmol), T4DNAligase1 μ L, spends the night 4 DEG C of ligations.

(6) product conversion DH5 α is connected

Under aseptic condition, in the DH5 α competent cell of 100 μ L, add 10 μ L connect product, ice bath 30min, then 42 DEG C of water-bath thermal shock 90s, rapid ice bath 1 ~ 2min after mixing.Often pipe adds 400 μ LLB substratum, 37 DEG C of recovery 60min; The centrifugal 6min of 5000rpm, abandons supernatant 400 μ L, on the remaining LB agar plate be applied to containing 100 μ g/mLAmp, cultivates 12 ~ 16h for 37 DEG C and occurs to single bacterium colony.

(7) PCR detects positive bacteria

Single bacterium colony on picking flat board in 3mL(containing the Amp of 100 μ g/mL) in LB substratum, 37 DEG C of 300rmp jolting overnight incubation.Get 1 μ L bacterium liquid to do template and carry out PCR detection.The company that checks order of the Wuhan section of holding up is sent to check order after detecting the positive.The sequencing result bacterium liquid consistent with GenBank sequence can direct enlarged culturing, extracts and removes intracellular toxin plasmid.

(8) ultrapure plasmid extraction

Adopt the extraction of Omega company Endo-freePlasmidMiniKitII test kit for the ultrapure plasmid of transfection.Single bacterium colony on picking flat board, be seeded to 3mL containing in corresponding antibiotic LB liquid nutrient medium, 37 DEG C of constant-temperature table overnight incubation, in the ratio enlarged culturing of 1:100; Inoculation object bacterium 37 DEG C of overnight incubation in 10 ~ 15mLLB.With 10mL centrifuge tube 5000g2min collection bacterium, stay the resuspended thalline of 1mL bacterium liquid.Re-suspension liquid is transferred in the centrifuge tube of 2mL.Clean nutrient solution to be abandoned after collection bacterium completes, add the SolutionI of 500 μ L, abundant resuspended thalline, repeatedly blow and beat (mixing, can not occur bacterial mass) with rifle head; Add the SolutionII of 500 μ L, slowly turn upside down 7 ~ 10 times, make the abundant cracking of thalline, room temperature is placed 2min and is become clarification (attention does not exceed 5min) to lysate, and SolutionII can not expose in atmosphere for a long time, after being finished, and cover lid immediately; Add the BufferN3 of 250 μ L precoolings, slowly turn upside down for several times, until generate white precipitate, with maximum centrifugal speed (being generally 12000r/min) 4 DEG C of centrifugal 10min; Carefully pipette the ETR adding 0.1 × volume in supernatant liquor to clean 1.5mL centrifuge tube, put upside down for several times mixing, hatch after 10min(adds ETR on ice, lysate becomes muddy, can become clarification, do not use the centrifuge tube of 2mL on ice after hatching).42 DEG C, 5min, lysate is again muddy, and 12000g3min, ETR will form the precipitation of blue layer at the bottom of pipe; Transfer upper strata aqueous phase, in 1 new 2mL centrifuge tube, adds the dehydrated alcohol of 0.5 × volume, slowly mixes 6 ~ 7 times, incubated at room 1 ~ 2min; Shift 700 μ L mixed solutions in HiBindTMDNAMiniColumnII and 2mL centrifuge tube, under room temperature, 10000g1min is centrifugal, abandons out waste liquid, repeats this step; Add 500 μ LHB and clean pillar, under room temperature, 10000g1min is centrifugal, abandons out flow liquid (removing residual protein to pollute); Add 700 μ LDNAWashingBuffer, under room temperature, 10000g1min is centrifugal, abandons out flow liquid, and repetitive operation once; The centrifugal 3min of top speed, film in dry pillar, pillar is put into the centrifuge tube of a new 1.5mL, add 80 ~ 100 μ LEndotoxin-FreeElutionBuffer, the centrifugal 5min of maximum speed, obtains eukaryotic expression vector pIRES 2-EGFP-CD151 and pIRES2-EGFP-CD163 that can express CD151 and CD163 molecule.

Finally pIRES2-EGFP-CD151 and pIRES2-EGFP-CD163 is carried out double digestion with SalI and KpnI respectively, verify through agarose gel electrophoresis to endonuclease bamhi, result as shown in Figure 2, shows construction of recombinant plasmid success.Plasmid its concentration of nucleic acid-protein analysis-e/or determining extracted.

Embodiment 2 cell transfecting

When Marc145 cell or MA-104 cytogamy reach more than 85%, be inoculated in new culture dish, in 37 DEG C, 5%CO ₂cultivation 24 hours in incubator, when cytogamy to 70 ~ 80%, carrying out CD151(plasmid used according to Lipofectamine2000 transfection reagent box process specifications is pIRES2-EGFP-CD151), CD163(plasmid used is pIRES2-EGFP-CD163=1:1), CD151 and CD163(plasmid used is pIRES2-EGFP-CD151:pIRES2-EGFP-CD163=1:1) transfection procedure, change after 6h and have serum, without dual anti-DMEM substratum (DMEM+10% foetal calf serum (Gibco company)).After transfection 48h, start with G418 substratum (DMEM+10% foetal calf serum+G418(Gibco company)) screening.Process is: 1st ~ 3 days 300 μ g/mL, then continues screening by the concentration of 600 μ g/mL, until most necrocytosis, G418 concentration is changed into 800 μ g/mL and maintains screening 6 weeks.Limiting dilution method is adopted to reach 96 porocyte culture plates in acquisition cell, about 1, every hole cell, be used alternatingly regular growth growth medium (DMEM+10% foetal calf serum (Gibco company)+100IU penicillin/streptomycin) and G418(600 μ g/mL) culture medium culturing individual cells, grow single clone to it.Under fluorescent microscope, pick out optimum cell clone continue to cultivate, and constantly expand numerous clone (strain) to setting up stable transfection CD151, CD163 and CD151/CD163 cotransfection, the Marc145 cell of transfection CD151, CD163 and CD151/CD163 cotransfection picture under fluorescence inverted microscope as shown in Figure 3, cell after transfection all expresses strong green fluorescence, shows that cell can stably express target protein.

The expression of embodiment 3 virus receptor in Marc145 cell

(1) cell cultures: cell culture processes cultivates the Marc145 cell of transfection CD151, CD163, CD151/CD163 and untransfected respectively routinely.In Bechtop, open six orifice plates, place sterile cover slip; Cell suspension is dropped on cover glass, be placed in CO ₂concentration is be cultured to cell attachment (about 2h) in 37 DEG C in the incubator of 5%; Add 2mL cell culture medium (DMEM+10% foetal calf serum (Gibco company)+100IU penicillin/streptomycin) and continue to cultivate about 6h; Remove substratum, with PBS(pH7.4) wash 3 times, each 5min.

(2) cell is fixed: 4% paraformaldehyde fixes 30min, and PBS washes 3 times, each 5min.

(3) cell rupture of membranes: creep plate slightly dries rear group and changes the position that is evenly distributed at cover glass intermediate cell of pen and draw a circle (preventing antibody from flowing away), add 50 ~ 100 μ L rupture of membranes working fluids (BD company), incubated at room 10min, PBS(pH7.4) wash 3 times, each 5min.

(4) primary antibodie is added: remove PBS, respectively with primary antibodie (all purchased from Abcam company) (rabbit anti-CD163,1:50 that PBS has diluted by a certain percentage; Rabbit anti-CD151,1:50; Rabbit anti-CD 69,1:50; Rabbit AntiCD3 McAb 4,1:100; Little mouse-anti CD44,1:100; Little mouse-anti CD90,1:100; Rabbit anti-vimentin, 1:100) cover tissue; Creep plate lies against 4 DEG C of overnight incubation in refrigerator.

(5) add two to resist: creep plate PBS(pH7.4) wash 3 times, each 5min; The fluorescence two dripping kind corresponding to primary antibodie after removing PBS in circle resists (all purchased from Abcam company), and (cy3 marks goat antirabbit 1:100; Cy3 marks goat anti-mouse 1:100) cover tissue, lucifuge incubated at room 50min.

(6) DAPI redyes nucleus: creep plate PBS(pH7.4) wash 3 times, each 5min; In circle, DAPI dye liquor is dripped, lucifuge incubated at room 10min after removing PBS.

(7) mounting: creep plate PBS(pH7.4) wash 3 times, each 5min; By anti-fluorescent quenching mountant, slide is locked in mounting on slide glass by having one of cell to face down after creep plate slightly dries.

(8) microscopy is taken pictures: cut into slices and to observe under Nikon inverted fluorescence microscope and to gather image, as shown in Figure 4, show that the cell after transfection can the corresponding receptor protein of overexpression.

The cell of embodiment 4 overexpression PRRS virus acceptor is improving the application in PRRS virus target cell infection titre and pig blue-ear disease vaccine production

(1) cell: the Marc145 cell of overexpression PRRS virus acceptor CD151, CD163 and CD151/CD163 coexpression or MA-104 cell.

(2) PRRS virus: JXA1-R(JXA1) strain.

(3) cell growth medium: the DMEM(Beijing Qingdatianyi Bioisystech Co., Ltd containing volumetric concentration being 8% calf serum).

(4) cell maintenance medium: the DMEM(Beijing Qingdatianyi Bioisystech Co., Ltd containing volumetric concentration being 2% calf serum).

(5) cell cultures: conveniently cell culture processes cell growth medium carries out cell and expands numerous cultivation (compiling " principle of vitro culture and technology " (publication of Science Press's calendar year 2001) with reference to Xue Qing philanthropist); The cell of enlarged culturing is inoculated in rolling bottle and cultivates, control rotating speed 10 turns/hour.

(6) viral proliferation: get desired number, cell fraction of coverage reach more than 80% cell spinner bottle, remove cell growth medium, according to 0.001MOI dose inoculation PRRS virus, add cell maintenance medium, at 37 DEG C continue cultivate.

(7) receive poison: examine under a microscope the cytopathy caused by virus, until gather in the crops virus liquid when it reaches more than 80%, fill into subsequently not containing the cell maintenance medium of virus, after 12h, receive poison again, repeatedly receive poison 5 times; TCID50 value (table 2) is calculated by Reed-Muench method.The virus liquid of results is placed in 2 ~ 8 DEG C, is kept at-20 DEG C.

(8) prepare vaccine: 1. living vaccine (JXA1-R strain): be the sucrose skimming milk lyophilized vaccine that 1:1 adds that mass concentration is 5% by the virus liquid of above-mentioned results by the volume ratio of virus liquid and lyophilized vaccine, make freeze-dried live vaccine.2. deactivation vaccine (JXA1 strain): by concentrated 10 times of the ultra-filtration membrane bag of the virus liquid 100KD molecular weight cut-off of above-mentioned results, the volume ratio of inactivator and virus liquid by 1:2000 add mass concentration be 37% formaldehyde solution carry out deactivation after, purifying antigen is obtained again through gel chromatography column Sepharose4FF column chromatography, add oily adjuvant in the ratio of antigen and oily adjuvant volume ratio 1:1, emulsification is mixed with inactivated vaccine (being formulated as 94%(V/V of oily adjuvant) white oil, 6%(V/V) Span-80,2%(g/V) aluminum stearate).

(9) vaccine potency inspection: 1. vaccine immunization: the dose inoculation piglet in 4 ~ 6 week age 10 of vaccine being pressed respectively 2mL/ head, establishes nonimmune control group 10 simultaneously.2. Serum Antibody Detection: gather porcine blood serum in latter 28 days with immunity before immunity, detect PRRS virus NAT.3. protest test: latter 28 days of immunity, to every pig muscle inoculation 3mL after diluting, continues breeding observing 21 days, slaughters and cut open inspection with PRRS virus (content is the virus liquid of 105.5TCID50/mL) 1:10.Detected result is in table 2.

Table 2 virus culture is tired and vaccine potency detected result

As can be seen from table in result, the present invention can express the cell of PRRS virus acceptor, compared with ordinary cells of the same type, cultivate on viral tiring, the former comparatively the latter exceed 2 ~ 5 times; On vaccine potency, the former with the latter is suitable.Total appraisal, the former production efficiency is higher than the latter 2 ~ 5 times.

Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.

SEQUENCELISTING

<110> Wuhan Inst of Veterinary Science

<120> mono-kind improves method and the application thereof of PRRS virus target cell infection titre

<130>1

<160>4

<170>PatentInversion3.5

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<211>29

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<220>

<223>CD151-F

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cggtcgacatgggcgaattcggcgagaag29

<210>2

<211>30

<212>DNA

<213>ArtificialSequence

<220>

<223>CD151-R

<400>2

cggggtacctcagtagtgctccagcttcag30

<210>3

<211>30

<212>DNA

<213>ArtificialSequence

<220>

<223>CD163-F

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cggtcgacatggtgctacttgaagactctg30

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cggggtacctcattgtacttcagagtggtc30

Claims

1. improve a method for PRRS virus (Porcinereproductiveandrespiratorysyndromevirus) target cell infection titre, it is characterized in that comprising the steps: with cell growth medium cultivate genetically modified after can the cell of overexpression PRRS virus acceptor; When cell fraction of coverage reaches more than 80%, remove cell growth medium, Pigs Inoculated reproductive and respiratory syndrome virus JXA1-R, add cell maintenance medium and continue to cultivate; When the cytopathy that virus causes reaches more than 80%, gather in the crops virus liquid, fill into subsequently not containing the cell maintenance medium of virus, after 12h, receive poison again, so repeatedly receive poison 3 ~ 5 times;

Described genetically modified after can the cell of overexpression PRRS virus acceptor be can overexpression CD151 molecule, CD163 molecule or co expression CD151 and CD163 molecule Marc145 or MA-104 cell, it is prepared by the method comprising following steps: be connected on eukaryotic expression vector pIRES 2-EGFP by coding CD151 molecule and/or CD163 molecular gene fragment, be transfected in Marc145 cell or MA-104 cell again, obtained the cell of overexpression PRRS virus acceptor by G418 and the screening of single cell clone culture method; Wherein, the primer of CD151, CD163 molecular gene fragment that increases is:

CD151-F：cgGTCGACATGGGCGAATTCGGCGAGAAG，

CD151-R：cggGGTACCTCAGTAGTGCTCCAGCTTCAG；

CD163-F：cgGTCGACATGGTGCTACTTGAAGACTCTG，

CD163-R：cggGGTACCTCATTGTACTTCAGAGTGGTC。

2. the method for raising PRRS virus target cell infection titre according to claim 1, is characterized in that: the dosage of described PRRS virus inoculation is 0.0005 ~ 0.002MOI.

3. the application of method in pig blue-ear disease vaccine is produced of the raising PRRS virus target cell infection titre described in claim 1 or 2.

4. produce the method for pig blue-ear disease vaccine for one kind, after it is characterized in that comprising the steps: to collect virus liquid according to the method for the raising PRRS virus target cell infection titre described in claim 1 or 2, virus liquid is mixed with lyophilized vaccine, makes freeze-dried live vaccine; Or by the ultra-filtration membrane bag concentrated more than 10 times of virus liquid with 100KD molecular weight cut-off, mixed with concentrated virus liquid by inactivator after carrying out deactivation, then obtain purifying antigen with gel chromatography column column chromatography, by antigen and the mixing of oily adjuvant, emulsification is mixed with inactivated vaccine.

5. the method for production pig blue-ear disease vaccine according to claim 4, is characterized in that: described lyophilized vaccine to be mass concentration be 5% sucrose skimming milk; Described inactivator to be mass concentration be 37% formaldehyde solution; Described oily adjuvant 94% white oil, 6%Span-80 and 2% aluminum stearate preparation.

6. the method for production pig blue-ear disease vaccine according to claim 4, is characterized in that: described virus liquid and the volume ratio of lyophilized vaccine are 1:0.5 ~ 2; Described inactivator is 1:1500 ~ 2500 with the volume ratio of concentrated virus liquid; The volume ratio of described antigen and oily adjuvant is 1:0.5 ~ 2.